Int. J. Mol. Sci. 2013, 14, 4545-4559; doi:10.3390/ijms14034545
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Cesium Inhibits Plant Growth through Jasmonate Signaling in
Arabidopsis thaliana
Eri Adams, Parisa Abdollahi and Ryoung Shin *
RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045,
Japan; E-Mails: [email protected] (E.A.); [email protected] (P.A.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +81-45-503-9577; Fax: +81-45-503-9650.
Received: 25 December 2012; in revised form: 25 January 2013 / Accepted: 6 February 2013 /
Published: 25 February 2013
Abstract: It has been suggested that cesium is absorbed from the soil through potassium
uptake machineries in plants; however, not much is known about perception mechanism
and downstream response. Here, we report that the jasmonate pathway is required in plant
response to cesium. Jasmonate biosynthesis mutant aos and jasmonate-insensitive mutant
coi1-16 show clear resistance to root growth inhibition caused by cesium. However, the
potassium and cesium contents in these mutants are comparable to wild-type plants,
indicating that jasmonate biosynthesis and signaling are not involved in cesium uptake, but
involved in cesium perception. Cesium induces expression of a high-affinity potassium
transporter gene HAK5 and reduces potassium content in the plant body, suggesting a
competitive nature of potassium and cesium uptake in plants. It has also been found
that cesium-induced HAK5 expression is antagonized by exogenous application of
methyl-jasmonate. Taken together, it has been indicated that cesium inhibits plant growth
via induction of the jasmonate pathway and likely modifies potassium uptake machineries.
Keywords: cesium; potassium; Arabidopsis thaliana; jasmonate; HAK5
1. Introduction
Cesium (Cs+) is a Group I alkali metal, which exists naturally at very low concentrations in the soil.
Cs+ has no known beneficial function in plants; however, it can, at high concentrations, cause toxicity,
observed as growth inhibition (reviewed in [1]). Thus far, Cs+ uptake and response in plants have not
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been thoroughly studied. However, the accident at the Fukushima nuclear power plant following the
disaster in 2011 in Japan highlighted the importance of understanding the mechanism of cesium uptake
in plants to improve phytoremediation efficiency for radiocesium. Radiocesium, 134Cs and 137Cs,
produced from anthropogenic sources are of environmental and health concerns, since they are rapidly
incorporated into the food chain, emit β and γ radiation and have relatively long half-lives (reviewed
in [1]). The potential of plants to decontaminate radiocesium from soil was also focused after the
accident in Chernobyl [2,3]. Earlier studies have suggested that Cs+ may be absorbed by plants in a
competitive fashion with potassium (K+), which belongs to the same alkali metal group and shares
similar chemical properties with Cs+. The first report that plants accumulate Cs+ through the same
uptake mechanism as K+ was published as early as 1941 [4], and many other studies reinforced this
view (reviewed in [1]). A theoretical modeling predicted that voltage-insensitive cation channels
(VICCs) predominantly mediate Cs+ uptake under the K+ sufficient conditions and K+ uptake permeases
(KUPs) significantly contribute under the K+ deficient conditions [1]. In Arabidopsis thaliana, some
members of cyclic nucleotide gated channels (CNGCs), which belong to VICCs, such as CNGC1,
CNGC2, CNGC4 and CNGC10, were predicted to form K+ channels and, thus, suggested as being
involved in Cs+ uptake, also [5–8].
The first K+ channel identified in plants is KAT1 from Arabidopsis, which functions at high K+
concentrations as a voltage-dependent, low-affinity inward rectifying channel [9]. The major K+
channel functioning in the roots of Arabidopsis under the K+ sufficient conditions is assumed to be
AKT1 [10]. However, the contribution of these channels to Cs+ uptake is small or negligible [9,11]. In
response to K+ deficiency, expression of high-affinity K+ transporters, members of the KUP family, are
induced, such as high affinity K+ transporter5 (HAK5) and KUP3 [12,13]. HAK5 has been reported to
be involved in Cs+ uptake in planta, consequently resulting in higher Cs+ accumulation under the K+
deficient conditions [12]. Another K+ transporter, KUP9, from Arabidopsis was also shown to
transport Cs+ when expressed in a K+ transport-deficient mutant of Escherichia coli [14].
There have been a few reports pointing out interaction between K+ deficiency response and
phytohormone signaling. Expression of ethylene biosynthesis genes and ethylene production are
reported to be induced in response to K+ deficiency and, subsequently, activate reactive oxygen species
production [15]. Expression of jasmonate (JA) biosynthesis genes is also induced in response to K+
deficiency and confers tolerance to insects [16–18]. Cytokinins, by contrast, decrease under the K+
deficient conditions and allow downstream response for adaptation to take place [19]. Auxin-related
mutants were found to be impaired in K+ deficiency-induced lateral root growth [20]. If Cs+ were to
cause similar effects as K+ deficiency in plants, it is possible that Cs+ response is regulated through
phytohormone pathways.
In this study, JA biosynthesis and signaling were demonstrated to be required in plant response to
+
Cs . JAs are a class of phytohormone that are involved in various physiological processes, such as
growth inhibition, fertility, senescence and defense against biotic and abiotic stresses [21]. JAs are
biosynthesized from linolenic acid through a series of steps, including allene oxide synthase
(AOS) [22], and recognized by a receptor complex which is composed of an E3 ubiquitin ligase, SCF
(SKP1, CDC53p/CUL1 F-box protein) complex. An active form of JAs, the jasmonyl-isoleucine
conjugate (JA–Ile), directly binds to the F-box protein in the receptor complex, CORONATINE
INSENSITIVE1 (COI1), and SCFCOI1 targets the negative regulators for proteolysis to activate
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downstream JA signaling in Arabidopsis [23–27]. Therefore, mutations in COI1 can render the plants
insensitive to JAs [28,29].
Here, we investigated the relationship between JA biosynthesis/signaling and Cs+ response in
Arabidopsis in order to understand the fundamental mechanism of plant response to Cs+. Our studies
revealed the induction of JA biosynthesis/signaling and consequent growth inhibition in response to Cs+.
However, JA signaling was not apparently involved in Cs+ uptake under the K+ sufficient conditions.
Meanwhile, induction of HAK5 was demonstrated in response to Cs+, suggesting the involvement of
K+ uptake machineries in Cs+ uptake. The possibility of JA signaling casting an antagonistic effect on
HAK5 expression was also implied. In this report, plant response to Cs+ is dissected.
2. Results
2.1. JA-Related Mutants Are Less Responsive to Cs+-Induced Growth Inhibition
In order to understand the mechanism of Cs+ response in plants, phytohormone-related mutants in
Arabidopsis thaliana were tested on 0.5 mM KCl with or without 0.3 mM CsCl. This condition was
chosen as a “stringent condition” for monitoring strong Cs+ resistance phenotype. In this condition,
growth of Col-0 plants (wild-type) was severely retarded by the addition of CsCl (Figure 1A).
However, a JA biosynthesis mutant aos [30,31] and a JA-insensitive mutant coi1-16 [29] showed
significantly less response to Cs+-induced root growth retardation (p < 0.001, Figure 1). A mutant,
which produces less bioactive JA-Ile and shows mild JA-insensitive phenotype, jasmonate resistant1
(jar1-1) [32], also showed less response to Cs+ in terms of root growth inhibition (p < 0.001, Table
S1). Among them, the stronger phenotype of aos corresponds with the fact that the aos mutant is a
knockout line [30]. The coi1-16 and jar1-1 mutants are known to be “leaky” [29,33], and this might be
why their resistance to Cs+ was also compromised. Also, other phytohormone-related mutants were
found to be less responsive to Cs+, such as an auxin-insensitive mutant, auxin resistant1 (aux1-7) [34]
(p < 0.001), and, to a lesser extent (p < 0.01), a trans-zeatin-type cytokinin biosynthesis quadruple
mutant, isopentenyltransferase1,3,5,7 (ipt1,3,5,7) [35] and a cytokinin-insensitive double mutant
histidine kinase2;3 (ahk2;3) [36] (Table S1). An ethylene-insensitive mutant, ethylene insensitive2,
(ein2-1) [37], but not ethylene response sensor1 (ers1-1) [38], was marginally less responsive to Cs+
(p < 0.05, Table S1). A GA-related DELLA double mutant, repressor of ga;gibberellic acid insensitive
(rga-24;gai-t6) [39], showed increased growth % in response to Cs+ compared to its wild-type, Ler
(p < 0.001, Table S1). This could be due to the absence of growth inhibitor proteins in the mutant [39],
enabling the resistance to Cs+-induced growth inhibition, but further analysis is required for
confirmation. The relationship between Cs+ response and JA signaling was further investigated.
2.2. Resistance of JA-Related Mutants to Cs+ Was Not Due to Less Uptake of Cs+
JA-related mutants were found to be less responsive to Cs+-induced growth inhibition compared to
wild-type. One explanation could be that JA-related mutants might be impaired in Cs+ uptake. To test
this possibility, K+ and Cs+ contents were measured in wild-type, aos and coi1-16 treated with or
without 0.3 mM CsCl under the K+ sufficient condition (1.75 mM KCl) for seven days. Reduced K+
contents were observed upon Cs+ treatment (p < 0.05, Figure 2A). However, there was no statistically
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significant difference observed between wild-type and mutants for both K+ and Cs+ contents,
regardless of Cs+ treatment (Figure 2). This suggests that the JA pathway is not involved in the uptake
of Cs+ or K+ in this condition. More K+ and Cs+ were accumulated per mg dry weight at the later stage
of growth (day 12), but the pattern was maintained (Figure S1).
Figure 1. (A) Phenotype of Col-0 (wild type), aos and coi1-16 grown on 0.5 mM KCl with
or without 0.3 mM CsCl for seven days. (B) Primary root % growth on 0.3 mM CsCl
compared to the control condition. Primary root lengths of the seedlings treated with or
without cesium were measured, and the % growth was calculated for Col-0 (black bar), aos
(crisscross bar) and coi1-16 (white bar). Error bars indicate standard error (n ≥ 20).
Statistically significant differences (p < 0.001) compared to Col-0 are indicated as asterisks.
(A)
(B)
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Figure 2. (A) K+ contents in Col-0, aos and coi1-16 grown on 1.75 mM KCl with
(white bars) or without (black bars) 0.3 mM CsCl for seven days. (B) Cs+ contents in Col-0,
aos and coi1-16 grown on 1.75 mM KCl with (dotted bars) or without 0.3 mM CsCl for
seven days. Error bars indicate standard error for four biological replicates. Each sample
contained more than 15 seedlings. b.d.: below the detection limit. Alphabetical letters show
statistical differences (p < 0.05).
(A)
(B)
2.3. Cs+ Induces JA Signaling as well as HAK5 Expression
To further analyze the interplay between Cs+ perception and JA signaling, expression of the marker
genes for JA signaling, PDF1.2 and VSP2 [40–42], was investigated in response to Cs+. Both PDF1.2
and VSP2 levels were increased upon Cs+ treatment under the K+ sufficient condition, especially in the
shoots (p < 0.05, Figure 3), suggesting the induction of the general JA pathway by Cs+. This induction
was absent in the aos mutant (Figure S2). There was no additive effect of Cs+ on methyl jasmonate
(MeJA)-induced VSP2 expression (Figure 3).
It has been reported that high-affinity K+ transporter, HAK5, is not only involved in K+ uptake, but
also in Cs+ uptake [12]. The expression of HAK5 is sharply induced in response to K+ deficiency [12],
and there are some indications that Cs+ may cause K+ deficiency in plants. Therefore, we analyzed the
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transcript levels of HAK5 in response to Cs+ under the K+ sufficient condition. Expression of HAK5
was, indeed, induced in response to Cs+, and this induction also occurred in JA-related mutants, aos
and coi1-16 (p < 0.05, Figure 4).
Figure 3. (A) Gene expression of PDF1.2 in Col-0 shoots and (B) roots, and (C) gene
expression of VSP2 in Col-0 shoots and (D) roots, grown on 1.75 mM KCl with or without
0.3 mM CsCl and 2.5 mM methyl jasmonate (MeJA) for 11 days. Values are log2 ratios
relative to the control. Error bars indicate standard error for three technical replicates. Each
sample contained more than 15 seedlings, and the experiment was repeated three times,
one of which is presented. Alphabetical letters show statistical differences (p < 0.05).
(A)
(B)
(C)
(D)
2.4. JA Antagonizes Cs+-Induced HAK5 Expression
Since HAK5 was found to be induced in response to Cs+, spatial localization of HAK5 was
investigated. Transgenic plants carrying the HAK5 promoter fused to the luciferase reporter construct
(HAK5promoter::LUC) were used, and their activities are known to be induced in K+ deficiency [15].
In this study, low levels of luciferase activity were visible at root tips of untreated plants under the K+
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sufficient condition (Figure 5). Upon Cs+ treatment, strong induction of luciferase activity was
observed in the roots, and this induction was abolished by the addition of MeJA (Figure 5). The
chemiluminescence intensity of these images and quantification of HAK5 transcripts in wild-type roots
treated with Cs+ and MeJA are shown in Table S2 and Figure S3, respectively. Chemiluminescence
images of HAK5promoter::LUC plants grown on 0.5 mM KCl with or without Cs+ are also presented
in Figure S4.
Figure 4. Gene expression of HAK5 in Col-0 (black bar), aos (crisscross bar) and coi1-16
(white bar) grown on 1.75 mM KCl with or without 0.3 mM CsCl for seven days. Values
are log2 ratios relative to the controls. Error bars indicate standard error for three technical
replicates. Each sample contained more than 15 seedlings, and the experiment was repeated
three times, one of which is presented. Statistically significant differences (p < 0.05)
compared to each non-treated sample are indicated as asterisks.
Figure 5. Chemiluminescence imaging of HAK5promoter::LUC plants grown on
1.75 mM KCl with or without 0.3 mM CsCl and 2.5 mM MeJA for 11 days. Pseudo color
represents the intensity of chemiluminescence.
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3. Discussion
Cs+ has no known nutritional or other function in plants. Thus far, scarcely any knowledge has been
gained on how plants absorb Cs+ from the soil, how it is transported inside the plant body, and what is
the plant response. However, an urgent need for improved phytoremediation methods for radioactive
Cs+ has urged scientists to better understand the mechanism of Cs+ uptake, transport and perception in
plants. In Arabidopsis, a possible mechanism for tolerance to heavy metals, such as arsenic, mercury
and cadmium, has been reported [43,44], but whether the same tolerance mechanism applies for Cs+ is
not yet known. A T-DNA insertion mutant which is impaired in Cs+ uptake has been isolated;
however, the identity of the gene disturbed is not revealed [45].
The similarity of the nature between Cs+ and K+ and the involvement of phytohormone pathways in
K+ deficiency response provoked us to investigate the relationship between phytohormone pathways
and Cs+ response. For this purpose, various phytohormone-related mutants in Arabidopsis thaliana
were analyzed for their response to Cs+. It has been reported that K+ deficiency response is observed
under 100 μM K+ concentrations in Arabidopsis [12]. Therefore, we used the 0.5 mM KCl condition,
which is well above the threshold for “K+ deficient conditions”, but lower than what is generally taken
as “K+ sufficient conditions” (1.75 mM KCl) [12], to create a “stringent condition” in selecting strong
phenotype mutants for Cs+. Of those mutants tested, JA-related mutants, aos, coi1-16 and jar1-1,
showed strong resistance to Cs+-induced growth inhibition. Our data suggest that the JA pathway, and
possibly some other phytohormone pathways, is involved in plant response to Cs+. This is the first
report, as far as we are aware, to demonstrate the interaction between Cs+ response and the JA pathway
in plants. Some phytohormone pathways, including ethylene, auxin, cytokinin and JA, have been
reported to be involved in response to K+ deficiency [15–20]; therefore, in the future, it will be
interesting to investigate the relationship of the Cs+-induced JA pathway with K+ deficiency.
Although aos and coi1-16 were less responsive to Cs+, this was not because they accumulated less
Cs+. Cs+ contents, as well as K+ contents, were comparable to those of wild-type plants, suggesting that
the JA pathway is involved, at least partly, in the perception of Cs+, not in uptake. Classic downstream
marker genes of the JA pathway, PDF1.2 and VSP2, represent two distinct branches of the signaling
pathway [40–42]. Under the K+ sufficient condition, both of these marker genes were found to be
increased in response to Cs+, especially in the aerial parts. This induction was not observed in the aos
mutant, indicating that Cs+ induces JA biosynthesis and downstream signaling.
It was also interesting to observe that K+ contents were reduced upon Cs+ treatment under the K+
sufficient condition. The competitive nature between Cs+ and K+ accumulation in Arabidopsis was
demonstrated in a previous report [46]. Although Cs+ has been used as a classic pharmacological
inhibitor of K+ channels [47], our findings might also indicate that Cs+ could have been absorbed
through the K+ uptake mechanism in a competitive manner. There are a few reports that suggest the
possibility of K+ transporters/channels absorbing Cs+ in plants, including a low-affinity inward-rectifying
channel in Arabidopsis, KAT1 [9], a high-affinity K+ transporter, HAK5 [12,48], and another K+
transporter, KUP9 [14]. In this study, we indicated that the transcript levels of HAK5 were
increased in response to Cs+ under the K+ sufficient condition. The reporter construct study using
HAK5promoter::LUC showed that its promoter activity was particularly notable in the actively
growing part of the roots, and this pattern was similar to that of the K+-starved plants [15]. These
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findings, together with the results from the K+/Cs+ content study, agree with the hypothesis that Cs+ is
absorbed through K+ uptake machinery in a competitive fashion.
JA treatment, on the other hand, was found to abolish Cs+-induced HAK5 expression in the reporter
construct study, suggesting an antagonistic effect of JA on HAK5 expression. Since Cs+ seemingly
induces both JA biosynthesis/signaling and HAK5 expression, this antagonism may function as a
negative feedback mechanism to fine-tune Cs+ response in plants.
A summary of our findings on Cs+-induced growth inhibition and gene expression is shown as a
model pathway in Figure 6. Cs+ induces JA biosynthesis and the downstream signaling pathway in
Arabidopsis. This is probably the major pathway for Cs+-induced growth inhibition of roots, since a
mutant incapable of producing JAs hardly shows any reduction in root length in response to Cs+. In
parallel, Cs+ induces HAK5, a reporter gene for K+ deficiency [49]. Further studies are required to
elucidate whether Cs+ treatment causes K+ deficiency to induce HAK5 expression in plants. It will be
interesting to test plant response to Cs+ under various K+ conditions, since plants are expected to
exhibit different K+ uptake mechanisms, according to K+ availability. It was also indicated that the JA
pathway might antagonize HAK5 expression. The mechanism in which this antagonism occurs awaits
elaboration; however, it is possible that one of APETALA2/Ethylene Response Factor (AP2/ERF)
transcription factors induced by the JA signaling pathway binds to the GCC-box of the HAK5 promoter
to regulate its expression. HAK5 has been reported to be regulated by an ERF/AP2 transcription factor,
RAP2.11, in response to K+ deficiency [50]. Meanwhile, JA-inducible AP2/ERFs are known to
regulate expression of a series of GCC-box-containing genes, such as PDF1.2, through direct binding
to the GCC-boxes of the promoter region [51–53]. Recently, involvement of cytokinin in K+
deficiency response and HAK5 expression has been reported [19]. It will be also interesting to further
investigate the involvement of biosynthesis and the signaling of cytokinin and other phytohormones in
Cs+ response in plants.
Here, we presented the first report that demonstrated the interplay between Cs+ response and JA
signaling in plants. It was also highlighted that Cs+ uptake, Cs+-induced growth inhibition and gene
expression were apparently regulated through different mechanisms. Elucidation of the fine balance
between K+ and Cs+ to regulate plant response and growth is awaited.
4. Experimental Section
4.1. Plant Material and Growth Conditions
The Arabidopsis thaliana (L.) Heynh. accession Col-0 and Ler were used as wild-types.
HAK5promoter::LUC was developed in the lab [15]. aos [30,31], ein2-1 [37] and jar1-1 [32] are
NASC stocks (Nottingham Arabidopsis Stock Centre, Loughborough, UK). coi1-16 [29], ipt1,3,5,7 [35],
ahk2;3 [36], ers1-1 [38], aux1-7 [34] and rga-24;gai-t6 [39] were gifts from Dr. Turner, Dr. Kakimoto,
Dr. Wen, Dr. Schachtman and Dr. Harberd. Seeds were surface-sterilized with 70% (v/v) ethanol and
0.05% (v/v) Triton X-100 and sown on media, indicated below. For the growth assay, media contained
0.5 mM KCl, 2 mM Ca(NO3)2, 0.5 mM phosphoric acid, 0.75 mM MgSO4, 50 μM H3BO3, 10 μM
MnCl, 2 μM ZnSO4, 1.5 μM CuSO4, 0.075 μM NH4Mo7O24 and 74 μM Fe-EDTA, pH 5.8, with
Ca(OH)2, 1% (w/v) sucrose and 0.5% (w/v) SeaKem agarose (Cambrex, Rockland, ME, USA)
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supplemented with or without 0.3 mM CsCl and for the rest of the study, media contained 1.75 mM
KCl, 2 mM Ca(NO3)2, 0.5 mM phosphoric acid, 0.75 mM MgSO4, 50 μM H3BO3, 10 μM MnCl, 2 μM
ZnSO4, 1.5 μM CuSO4, 0.075 μM NH4Mo7O24 and 74 μM Fe-EDTA, pH 5.8, with Ca(OH)2, 1% (w/v)
sucrose and 0.5% (w/v) SeaKem agarose (Cambrex) supplemented with or without 0.3 mM CsCl or
2.5 μM MeJA. After stratifying for 3 to 4 days at 4 °C, plants were placed in a vertical orientation in a
growth cabinet at 22 °C in a 16 h light/8 h dark photocycle with a light intensity of 70–90 μmol/m2/sec.
Figure 6. Model pathway. Cs+ induces the JA pathway and inhibits plant growth. Cs+ also
induces HAK5 expression. Whether this expression is due to Cs+-induced K+ deficiency or
the direct effect of Cs+ is not yet clear. An antagonistic effect of the JA pathway on HAK5
expression is also suggested.
4.2. Root Growth Assay
Seedlings grown for 7 days were removed from media, and the root lengths were measured directly
on a ruler wetted with 70% (v/v) glycerol. The relative % of root growth was calculated according to
the formula, y/x × 100, where x was the average of root lengths of untreated seedlings and y was the
root lengths of each Cs+-treated seedling and was averaged. One-way ANOVA with Dunnett’s
multiple comparison posttest (p < 0.05) was performed using Prism (GraphPad Software, La Jolla,
CA, USA) to determine the statistical significance.
4.3. Element Analysis
Seedlings grown for 7 days were washed in Milli-Q water, dried on a piece of paper towel, placed
in a paper envelope and dried in an oven at 65 °C for 3–4 days. Approximately 2 mg of dried samples
were extracted in 1 mL of 60% (v/v) HNO3 at 125 °C for 1 h, followed by 1 mL of 30% (v/v) H2O2 and
diluted with Milli-Q water to get a total volume of 10 mL. For K+ analysis, samples were further
diluted 10 times with 6% (v/v) HNO3. For Cs+ analysis, 0.1% (w/v) KCl was added to each sample and
standard solution to prevent ionization of Cs+, according to the manufacturer’s instructions
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(PerkinElmer, Waltham, MA, USA). K+ and Cs+ contents were measured on a flame atomic absorption
spectrometer AAnalyst 200 (PerkinElmer). Concentrations were calculated against each standard
curve, and one-way ANOVA with Bonferroni's multiple comparison posttest (p < 0.05) was performed
using Prism to determine the statistical significance.
4.4. qRT-PCR Analysis
Seedlings grown for 7 days (for HAK5 expression) or 11 days (for VSP2 and PDF1.2 expression)
were flash-frozen in liquid N2 and ground using a mixer mill. Total RNA was extracted, treated
with DNaseI (Invitrogen, Carlsbad, CA, USA) and synthesized into cDNA using SuperScript III
(Invitrogen). Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed using
THUNDERBIRD SYBR qPCR mix (TOYOBO, Osaka, Japan) and a Mx3000P qPCR system (Agilent
Technologies, Santa Clara, CA, USA). The amplification conditions were 95 °C for 15 s and 60 °C for
30 s. The cycle was repeated 40 times, preceded by 95 °C for 1 min and followed by a dissociation
program to create melting curves. Three technical replicates for each sample were run. The β-tubulin gene
(TUB2) was used as a reference gene. The primers used were as follows: HAK5 forward
5’-CGAGACGGACAAAGAAGAGGAACC and reverse 5’-CACGACCCTTCCCGACCTAATCT [49];
VSP2 forward 5’-CCTAAAGAACGACACCGTCA and reverse 5’-TCGGTCTTCTCTGTTCCGTA [54];
PDF1.2 forward 5’-TTGCTGCTTTCGACGCA and reverse TGTCCCACTTGGCTTCTCG [51];
and
TUB2
forward
5’-GCCAATCCGGTGCTGGTAACA
and
reverse
5’-CATACCAGATCCAGTTCCTCCTCCC [49]. One-way ANOVA with Bonferroni’s multiple
comparison posttest (p < 0.05) was performed using Prism to determine the statistical significance.
4.5. Luciferase Imaging
HAK5promoter::LUC grown for 11 days were sprayed with luciferin (Duchefa Biochemie,
Haarlem, The Netherlands) and kept in the dark for a few minutes prior to imaging. NightSHADE LB
985 (Berthold, Bad Wildbad, Germany) was used to image luciferase chemiluminescence.
5. Conclusions
In this study, we have determined that JA signaling is involved in Cs+ perception in terms of root
growth inhibition, but not in uptake of K+ and Cs+. This growth inhibition by Cs+ occurs presumably
through biosynthesis of JAs and consequent stimulation of the downstream JA signaling pathway. In
parallel, Cs+ induces expression of a high-affinity K+ transporter gene, HAK5, under the K+ sufficient
condition in a JA signaling-independent manner. However, exogenous application of MeJA inhibits
Cs+-induced expression of HAK5, suggesting a complicated “fine-tuning” regulation of Cs+ response
in plants.
Acknowledgments
Many thanks to Prof. John Turner (University of East Anglia) for providing coi1-16 seeds, Prof. Tatsuo
Kakimoto (Osaka University) for ipt1,3,5,7 and ahk2;3 seeds, Prof. Chi-Kuang Wen (Shanghai Institute for
Biological Science, Chinese Academy of Science) for ers1-1 seeds, Dr. Daniel Schachtman (Donald
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Danforth Plant Science Center) for aux1-7 seeds and Prof. Nicholas Harberd (University of Oxford) for
rga-24;gai-t6 seeds. This work was supported by funding from RIKEN and the Ministry of
Agriculture, Forestry and Fisheries of Japan (I2012101).
Conflict of Interest
The authors declare no conflict of interest.
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